HNF4A modulates glucocorticoid action in the liver

Summary The glucocorticoid receptor (GR) is a nuclear receptor critical to the regulation of energy metabolism and inflammation. The actions of GR are dependent on cell type and context. Here, we demonstrate the role of liver lineage-determining factor hepatocyte nuclear factor 4A (HNF4A) in defining liver specificity of GR action. In mouse liver, the HNF4A motif lies adjacent to the glucocorticoid response element (GRE) at GR binding sites within regions of open chromatin. In the absence of HNF4A, the liver GR cistrome is remodeled, with loss and gain of GR recruitment evident. Loss of chromatin accessibility at HNF4A-marked sites associates with loss of GR binding at weak GRE motifs. GR binding and chromatin accessibility are gained at sites characterized by strong GRE motifs, which show GR recruitment in non-liver tissues. The functional importance of these HNF4A-regulated GR sites is indicated by an altered transcriptional response to glucocorticoid treatment in the Hnf4a-null liver.

INTRODUCTION NR3C1, the glucocorticoid receptor (GR), is an almost ubiquitously expressed nuclear receptor. While there is evidence for rapid, non-genomic actions of glucocorticoids (Dallman, 2005;Kershaw et al., 2020), the chief outcomes of GR activation occur through its nuclear activity. GR binds the genome through the glucocorticoid response element (GRE) motif, which comprises two palindromic hexamers separated by a 3 bp spacer (AGAACANNNTGTTCT). Formation of higher-order GR structures (dimerization, tetramerization) is necessary for GR-mediated gene regulation (Johnson et al., 2021).
Upon ligand binding, GR is predominantly directed to sites on the genome where chromatin is already accessible (John et al., 2011), and this is dependent on cell type, with priming by C/EBPB (CCAAT-enhancer binding protein beta) particularly important in the liver . GR can also demonstrate pioneer function at sites of inaccessible chromatin (Johnson et al., 2018). While it is less clear what the determinants of binding are here, similarity of the GR-bound DNA sequence to the canonical GRE (''motif strength'') may play a role, with nucleosome-deplete sites demonstrating more degenerate GRE motifs (Johnson et al., 2018). Other studies have shown that active histone marks and presence of pioneer factors also play a role in dictating GR binding (McDowell et al., 2018). Following GR binding, gene activation-involving recruitment of coactivators and chromatin remodelers-occurs at sites of pre-established enhancer-promoter interactions, with the presence of GR increasing the frequency of productive interactions (D'Ippolito et al., 2018). In contrast, the mechanism by which GR downregulates gene expression remains an area of considerable debate, with evidence for protein-protein tethering, indirect mechanisms of action, and GR binding to negative or cryptic response elements presented (Escoter-Torres et al., 2020;Hudson et al., 2018;Miranda et al., 2013;Oh et al., 2017;Sacta et al., 2018;Surjit et al., 2011).
Surprisingly for a transcription factor which is so widely expressed, GR action is remarkably context specific. GR activity can be influenced both by metabolic and immune state (Goldstein et al., 2017;Quagliarini et al., 2019;Uhlenhaut et al., 2013). GR action is also highly tissue specific, with the GR cistrome showing limited overlap between different cell types (John et al., 2011). This is a property which is far from unique to GR, and has been well illustrated for other transcription factors from multiple classes, including the estrogen receptor (Gertz et al., 2013) and the core clock protein BMAL1 (Beytebiere et al., 2019). In vitro studies suggest that GR cell specificity is conferred by differential chromatin accessibility at distal enhancer sites, with GR binding in proximal promoter regions regulating genes that are ubiquitously glucocorticoid responsive (Love et al., 2017). In this in vivo study, we show the dominance of hepatocyte nuclear factor 4A (HNF4A), itself a nuclear receptor, in determining GR binding in mouse liver. We find the HNF4A motif to underlie sites of GR binding and, in Hnf4a-null liver, demonstrate loss of chromatin accessibility and GR binding at HNF4A-marked sites. We see emergence of non-liver-specific GR binding events at sites characterized by strong GRE motifs. We find evidence that this remodeling of the GR cistrome is of functional importance in shaping an altered transcriptomic response to glucocorticoids in the absence of HNF4A.

HNF4A motifs mark liver GR binding sites
We first mapped the hepatic GR cistrome by performing GR ChIP-seq on mouse liver collected 1 h after intraperitoneal injection of dexamethasone (DEX) at Zeitgeber time 6 (mice housed in 12 h:12 h light-dark cycles, ZT0 = lights on, n = 2) ( Figure 1A). As expected, DEX treatment resulted in substantial GR recruitment to the genome, with 20,064 peaks called over input (q < 0.01) in DEX-treated tissue.
We have previously shown that the same model of glucocorticoid treatment has a large effect on the liver transcriptome, with the expression of 1,709 genes being significantly altered (1,052 upregulated, 657 downregulated) (Table S1) (Caratti et al., 2018). We now observed pronounced enrichment of glucocorticoid-upregulated genes in relation to GR binding sites ( Figure 1B; hypergeometric test) (Briggs et al., 2021;Yang et al., 2019). Enrichment was seen at distances of 500 bp-500 kbp between transcription start sites of DEX-activated genes and GR ChIPseq peaks ( Figure 1B), implying both proximal and distal regulation. The noticeably weaker enrichment of glucocorticoid-downregulated genes supports the notion, reported by others (Jubb et al., 2016;Oh et al., 2017), that gene downregulation occurs by indirect means; however, we cannot exclude mechanisms, such as tethering, based on these data.
We proceeded to motif discovery analysis, and found the canonical GRE to be the most highly enriched known motif within GR binding sites, followed by the HNF4A motif ( Figure 1C; see also Mendeley data: https://doi.org/10.17632/k8d386ndz6.3). A motif most closely resembling the HNF4 motif had the lowest p value on de novo motif analysis ( Figure 1C), and was detected in 25.64% of GR peaks. As reported for other GR ChIP-seq studies (Hemmer et al., 2019;Lim et al., 2015), other motifs detected (at lower significance) included CEBP, PPAR, and HNF6 motifs. A similar pattern of motif enrichment was seen in GR ChIP-seq peaks called in vehicle-treated liver (n = 2) (5,831 peaks called over input), with the known GRE and HNF4A motifs being the most strongly enriched, and a HNF4-like motif detected de novo in 29.29% of peaks (Figures S1A and S1B). Interestingly, we still observed strong enrichment of DEX-upregulated genes at distances of 5-500 kbp from GR peaks in VEH liver ( Figure S1C), implying some pre-existing GR binding in association with glucocorticoid-responsive genes, likely reflecting the action of the endogenous GR ligand corticosterone.
We were interested to know how closely GRE and HNF4A motifs were situated at GR binding sites, as the distance between transcription factor (TF) motifs contributes to the likelihood of TFs co-occupying regulatory elements (Sö nmezer et al., 2021). Under GR peaks where we observed co-occurrence of HNF4A and GRE motifs, we saw a spread of inter-motif distances (Figure S1D), with 30-50 bp being most frequent, irrespective of whether motif calling was performed with high stringency settings (''strong'' GRE/HNF4A), or permitted some degeneracy (''weak''). DNA footprinting approaches (Sö nmezer et al., 2021) suggest that TF co-occupancy is most likely at inter-motif distances <20 bp, with a random distribution of co-occupancy events observed at inter-motif distances >70 bp. High levels of co-occupancy could suggest that binding is physically co-operative (Sö nmezer et al., 2021). When binned in this way, our data show that inter-motif GRE-HNF4A distances of 20-70 bp are most common ( Figure 1D), favoring co-occupancy of GR and HNF4A at regulatory elements, but not necessarily supporting physical co-operation between the two nuclear receptors.
To explore the importance of HNF4A via an independent approach, we performed ATAC-seq (assay for transposaseaccessible chromatin) on DEX-and VEH-treated mouse liver. We used HOMER to map the positions of all canonical GREs in the mouse genome (>137,000 locations). At GREs with a nearby HNF4A motif (within 20 bp, 3,281 sites; at 20-70 bp, 2,986 sites; at 70-150 bp, 4,872 sites), we observed stronger mean ATAC signal in DEX-treated liver than at GREs without a nearby HNF4A motif (>125,000 sites) ( Figure 1E), with signal strongest at 20-70 bp inter-motif distances. ATAC signals centered around HNF4A motifs (total >567,000 locations) echoed these data, being greatest at HNF4A motifs with a GRE motif within 20-70 bp ( Figure 1E). In VEH-treated liver, we observed the same patterns, albeit with an overall reduction in ATAC signal associated with GREs ( Figure S1E), likely reflecting the reduction of nuclear (and thus genomic) recruitment of GR in the absence of ligand activation. Therefore, in liver, chromatin accessibility at colocated GRE HNF4A motifs is greater than isolated motifs of GRE, or HNF4A alone, suggesting that HNF4A may play a role in determining chromatin accessibility at GR binding sites, and vice versa. Furthermore, integration with gene expression data indicates that co-located GRE and HNF4A motifs mark regulatory elements where GR acts to upregulate gene expression in the cis domain. This is in line with existing theories that GR binds the genome at pre-programmed, DNase-sensitive sites John et al., 2011;Praestholm et al., 2020), with glucocorticoid treatment augmenting GR action at these sites, and increasing the frequency of pre-established enhancer-promoter interactions (D'Ippolito et al., 2018).

HNF4A loss remodels the GR cistrome
We thus hypothesized that removing HNF4A would impact upon patterns of GR binding. To test this, we performed GR ChIP-seq on livers from 6-to 8-week-old Hnf4a fl/fl Alb Cre mice (Hayhurst et al., 2001) treated acutely with DEX, again at ZT6. Hnf4a fl/fl Alb Cre+/À mice are viable, but show hepatomegaly and hepatosteatosis from age 6 weeks, and increased mortality from age 8 weeks (Hayhurst et al., 2001). Nonetheless, they present a useful, widely used model (Fekry et al., 2018;Karagianni et al., 2020;Wu et al., 2020;Xu et al., 2021) to study how HNF4A regulates transcriptional activity in vivo, with loss of liver Hnf4a gene expression ( Figure S2A) and protein evident . We performed a differential binding analysis Smyth, 2015, 2016) [LWT]) and Hnf4a fl/fl Alb Cre+/À (liver knockout [LKO]) livers (n = 2/group). We employed an internal spike-in normalization strategy with D. melanogaster chromatin  to control for technical variation, and so increase confidence that results represented true genotype effects.
This approach detected 4,924 sites where GR binding was lost in LKO animals compared with LWT (FDR < 0.05), and 989 sites where GR binding was gained. Loss of HNF4A therefore led to substantial remodeling of the liver GR cistrome (Figures 2A and  2B). Lost and gained sites chiefly annotated to intergenic and intronic regions of the genome, suggesting that remodeling was affecting distal regulatory sites rather than proximal promoter re-gions ( Figure S2B). In keeping with previous work (Love et al., 2017), this supports the notion that tissue specificity of GR action may be largely conferred by distal enhancers, rather than proximal promoter binding.
To examine the distinctions between lost and gained GR sites in more detail, we first quantified the abundance of specific motifs. We observed that GR sites lost in LKO liver showed low abundance of the canonical GRE and high abundance of the HNF4A motif, while GR sites gained showed high abundance of the canonical GRE and low HNF4A motif abundance ( Figure 2C). These findings were recapitulated by motif discovery analysis ( Figure 2D; see also  (E) ATAC-seq coverage score (mean coverage from three biological replicates), in DEX-treated liver, around canonical GRE motifs with or without a HNF4A motif within specified distances (left panel), and around HNF4A motifs with or without a GRE motif within specified distances (right panel). See also Figure S1 and Table S1.
Cell Reports 39, 110697, April 19, 2022 3 Article ll OPEN ACCESS motif being the most strongly enriched in lost GR sites supports the specificity of the effect. On comparing the strength of GRE motifs in lost and gained sites (scored by similarity to the canonical GRE), we found lost GR sites to be predominantly characterized by weak GREs, while strong GREs characterized a population of gained sites ( Figure 2E). Within lost sites, cooccurrence of GRE and HNF4A motifs was most numerous at inter-motif distances of 20-70 bp ( Figure S2C), as we observed for the wider GR cistrome.  overlap of lost GR sites with HNF4A binding sites ( Figure 2F), suggesting that the HNF4A protein, in addition to the motif, can normally be found at these sites. Unsurprisingly, we saw almost no overlap of gained GR sites with the HNF4A cistrome. Therefore, in the absence of HNF4A, GR is no longer recruited to sites marked by the HNF4A motif (and HNF4A binding) and a weak GRE motif. Intriguingly, GR binding emerges at sites where it is not normally recruited, where strong GRE motifs are present (and where HNF4A is not found). This marked divergence between lost and gained sites points to this being a specific consequence of HNF4A loss, and not a downstream effect of the abnormal hepatic physiology of Hnf4a fl/fl Alb Cre+/À livers.
HNF4A-dependent GR sites demonstrate distinct patterns of chromatin accessibility and tissue GR recruitment Next, we sought to understand the normal chromatin state of GR sites lost or gained in the absence of HNF4A. We took advantage of published datasets for DNase hypersensitivity  and histone mark ChIP-seq (Armour et al., 2017;Shen et al., 2012) in naive mouse liver (i.e., the chromatin landscape that the GR encounters upon dexamethasone treatment) to quantify read coverage at lost and gained sites. We found that GR is lost at sites where, in liver, chromatin is normally DNase sensitive, and where higher levels of the histone marks H3K27ac and H3K4me1-associated with active/poised enhancers-are found. GR binding is gained at sites where chromatin is normally less DNase sensitive, and where the H3K27me3 mark (associated with inactive regions of heterochromatin) is stronger ( Figure 3A).
Given that HNF4A is a lineage-determining TF, we hypothesized that loss of HNF4A results in a GR cistrome less specific to the liver. We performed an unbiased comparison of lost and gained sites with TF cistromes, using the GIGGLE tool (Layer et al., 2018). We found lost sites to be normally bound by not only HNF4A itself, but by multiple other factors with important metabolic roles, with almost all of these cistromes being derived from liver/hepatocyte experiments ( Figure 3B; see also Mendeley data: https://doi.org/10.17632/k8d386ndz6.3). In marked contrast, gained sites were most numerously bound by GR (NR3C1) and other NR3 family members, but almost exclusively in non-liver tissues. Importantly, these tissues were diverse, and not simply non-hepatocyte cell types (e.g., inflammatory cells) that might be found within liver tissue. Interestingly, those liver GR cistromes that did show overlap with gained sites ( Figure 3B) were from a study where overexpression of a dominant negative form of C/EBP was used to disrupt the liver GR cistrome .
Thus, these findings suggest that HNF4A is necessary for directing GR binding at a population of liver-specific sites by means of maintaining open chromatin. The GREs at these HNF4A-dependent sites show degeneracy from the canonical AGAACANNNTGTTCT motif, but chromatin state is favorable toward GR binding, being marked by active histone modifications. It may be that other important regulators of liver energy metabolism are also recruited to these regions. By contrast, in the absence of HNF4A, GR is recruited to additional sites where chromatin is not normally accessible in liver, but where a strong GRE motif is found, and where GR is capable of binding in other tissues. Indeed, we observed significantly greater overlap between gained GR sites and GR cistromes in non-liver tissues (white adipose, macrophages, mammary gland), than between lost GR sites and non-liver GR cistromes ( Figures 3C and 3D).
In the absence of HNF4A, chromatin accessibility is remodeled at HNF4A-dependent GR sites To test the hypothesis that GR binding is determined by HNF4Adependent chromatin accessibility, we proceeded to ATAC-seq to map transposase-accessible sites in intact and Hnf4a-null liver (VEH and DEX treated), across a total of ten samples. As expected, ATAC-seq confirmed excision of Hnf4a exons 4 and 5 in Hnf4a fl/fl Alb Cre LKO mice ( Figure 4A). On performing differential accessibility analysis with csaw, we saw a stark distinction between LWT and LKO samples ( Figure 4B), indicating that HNF4A loss leads to substantial remodeling of the chromatin environment. In LKO mouse liver, compared with LWT, chromatin accessibility was significantly reduced (FDR < 0.05) at 16,103 sites in VEHtreated animals, and significantly increased at 14,531 sites (Figure 4C). In DEX-treated LKO animals, 17,400 sites showed reduced accessibility and 16,691 sites increased accessibility. We also saw that glucocorticoid treatment affected chromatin accessibility ( Figure 4B), with DEX treatment increasing accessibility at GRE-and CEBP-marked sites (Mendeley data: https:// doi.org/10.17632/k8d386ndz6.3).
We proceeded to more detailed motif analysis of sites where chromatin accessibility was impacted by Hnf4a deletion. Motif discovery found sites with greater accessibility in LWT mice (i.e., reduced in LKO) to be strongly enriched for the HNF4A motif ( Figures 4D and 4E; see also Mendeley data: https://doi.org/10. 17632/k8d386ndz6.3), while sites with greater accessibility in LKO mice were enriched for Forkhead box (FOX) motifs (FOXM1; Figure 4D; see also Mendeley data: https://doi.org/ 10.17632/k8d386ndz6.3). These data indicate that loss of HNF4A results in reduced chromatin accessibility primarily at HNF4A binding sites, and increased chromatin accessibility at sites marked by FOX factors.
We thus hypothesized that the remodeling of the GR cistrome seen with Hnf4a deletion chiefly reflects remodeling of the chromatin environment. Importantly, the chromatin remodeling we observed was of a different order of magnitude to the remodeling of the GR cistrome (>30,000 loci with altered ATAC-seq signal, compared with $5,000 loci with altered GR binding). This implies that the impact of HNF4A loss extends beyond the GR cistrome and, indeed, this is corroborated by recent work showing large changes in the BMAL1 cistrome with Hnf4a deletion (Qu et al., 2021).
On quantifying ATAC signal at sites of differential GR binding, chromatin accessibility positively correlated with GR binding ( Figure 4F); where GR binding is lost in LKO animals, we observed reduction of chromatin accessibility; where GR binding is gained, we saw increased chromatin accessibility. A total of 77% of lost GR sites (3,777 sites) directly overlapped with the 17,400 sites of significantly decreased chromatin accessibility; 57% of gained GR sites (568 sites) overlapped with the 16,691 sites of increased accessibility (the incomplete overlap of altered GR sites with altered accessibility sites likely results from false negatives and false positives in each group of sites). Supporting Cell Reports 39, 110697, April 19, 2022 5 Article ll OPEN ACCESS concurrent loss of HNF4A binding at the lost GR sites, we saw a marked alteration in the ATAC-seq signal ''footprint'' from LKO samples around HNF4A motifs ( Figure 4G).
Our GR ChIP-seq and ATAC-seq data together indicate, therefore, that HNF4A loss in Hnf4a fl/fl Alb Cre LKO liver re-duces accessibility of chromatin at sites usually marked by HNF4A, reducing GR recruitment to co-located weak GREs. Increased accessibility at FOX-marked sites, associated with strong GREs, increases GR recruitment to these loci. Datasets from non-liver tissues/cells plotted in blue, datasets from liver/hepatocytes plotted in red.
(D) Exemplar tracks showing GR ChIP-seq signal around the Tsc22d3 (Gilz), Uvrag, and Nrg4 loci in LWT and LKO liver, and in bone marrow-derived macrophages . Universal, macrophage-specific and liver-specific GR sites highlighted by arrows. The y axis is uniform within each panel.  Hnf4 f4 f4 f4 f4 f4 f4 f4 f4 f4 f4 f4 f4 f4 f4 f4 f4 f4 f4 f4 f4a a a a a a a a a a a a a a a a a a a

HNF4A loss remodels the glucocorticoid-responsive transcriptome
To examine the functional importance of this GR cistrome remodeling, we then performed liver RNA-seq to quantify gene expression in dexamethasone-and vehicle-treated Hnf4a fl/fl Alb Cre LWT and LKO mice (n = 3-4/group). Simply by studying differential gene expression in vehicle-treated mice, we saw a profound effect of HNF4A on the liver transcriptome ( Figure 5A), with the expression of >7,000 genes being different between genotypes. Genes with diminished expression in LKO mice were characterized by pathways of lipid, amino acid, and oxidative metabolism, while genes with increased expression in LKO mice were associated with non-liver pathways, such as Rho GTPase (intracellular actin dynamics) and cell-cycle pathways ( Figure 5B). This loss of liver identity with Hnf4a deletion echoes observations in models of non-alcoholic steatohepatitis where, interestingly, ATAC-seq analyses indicate loss of HNF4A activity (Loft et al., 2021). Importantly for our hypothesis, loss of HNF4A also altered the response to acute glucocorticoid treatment. By comparing gene expression in LKO and LWT mice treated with dexamethasone or vehicle, using R Bioconductor package stageR (Van den Berge et al., 2017), specifically designed to control FDR at the gene level, we found 1,908 genes where a significant genotype-treatment interaction was detected (adjusted p value < 0.05). Of these 1,908 genes, 633 showed a marked difference in response to DEX treatment between the two genotypes ( Figure 5C). Of note, these included important metabolic genes (e.g., Ppara, Gdf15, Gck), suggesting that HNF4A exerts a major impact on the liver metabolic response to glucocorticoids. Gdf15 expression, for example, is normally upregulated by glucocorticoid, an effect that is lost in the Hnf4a fl/fl Alb Cre+/À mice. Pathway analysis of DEX-upregulated genes with a treatment-genotype interaction (Tables S2 and S3) detected enrichment of cholesterol biosynthesis and steroid metabolism processes, suggesting that glucocorticoid regulation of these pathways might be under HNF4A control, specifically targeting these actions to the liver.
To determine whether these changes in transcriptomic response might directly relate to the remodeling of the GR cistrome, we looked for significant enrichment of genes of interest in the locale of lost and gained GR sites also marked by significant change in chromatin accessibility (3,777 lost sites, 568 gained sites). Specifically, we looked at the 1,908 genes where stageR detected a significant treatment-genotype interaction, and asked whether these were over-represented in proximity to these GR + ATAC sites ( Figure 5D). As control clusters, we took DEX-responsive genes where stageR did not detect such an interaction (3,797 genes) and randomly sampled 1,908 genes from this group, repeating this random sampling four times.
At GR + ATAC sites lost in the absence of HNF4A, at distances between 100 bp and 500 kbp, we saw strong enrichment of genes with a treatment-genotype interaction (e.g., Klf3 and Jun, Figures 5D and 5E). Interestingly, strong of genes without an interaction was also apparent, but at distances of 50-100 kbp from lost GR sites ( Figure 5D). It may be that a proportion of these sites are distal enhancers that make a redundant contribution to the regulation of the genes in question, or a contribution that is sufficiently small not to be detected in our RNA-seq analysis. Other, HNF4A-independent regulatory elements may exert more dominant control over these genes.
For GR + ATAC binding sites gained in LKO liver, we observed some genes with a treatment-genotype interaction (e.g., Slc2a4, Fos) to lie in proximity to gained sites ( Figure 5E), but did not observe strong enrichment at the genome-wide level ( Figure 5D), which may be a reflection of the small number of sites involved. Our results support the idea that loss of GR binding in Hnf4a-null liver contributes to the observed alteration in the transcriptional response to glucocorticoid treatment, and that the remodeling of the GR cistrome is of functional importance. While we are not able to definitively prove a cause-effect relationship, we see strong statistical associations across thousands of sites and thousands of genes. Gained GR sites may also alter the glucocorticoid response, but with an effect size that is masked by the large impact of Hnf4a deletion on the global transcriptome.
Given the emergence of GR binding and chromatin accessibility at sites marked by FOX TFs (Figures 2D and 4D), we specifically looked at expression of FOX factor genes ( Figure 5F). FOX proteins have been proposed to have pioneer function, and colocalize with steroid hormone receptors (Sanders et al., 2013;Zhang et al., 2005). Of note, we saw increased expression of Foxm1 (Hnf3) in LKO liver, compared with LWT. Altered FOXM1 activity may therefore contribute to the chromatin remodeling observed with HNF4A loss.
HNF6 has limited influence on liver GR action Finally, we were also interested to examine the influence of a hepatic lineage-determining factor from another family. We have found HNF4A deletion to have a substantial effect on GR binding, and others have demonstrated the importance of the bZIP TF C/EBPB . HNF6 emerged in our early analysis of motifs related to GR binding sites ( Figure 1C). HNF6 (Hnf6), is a lineage-determining factor that is part of the onecut family of TFs (Odom et al., 2004). Although its motif was enriched at hepatic GR binding sites ( Figure 1C), it was found at a smaller proportion of GR sites than the HNF4A motif, and its presence in the vicinity of GREs is not associated with the large increase in chromatin accessibility seen with the HNF4A motif ( Figure S3A).
We therefore hypothesized that HNF6 plays a more minor role in shaping liver GR action, although HNF6 is required for embryonic development and liver specification. We used a mouse model of postnatal liver Hnf6 deletion (its embryonic loss is lethal) ( Figure S3B). We found that this had only a small effect on the liver transcriptome under basal conditions ( Figure S3C), with a correspondingly minor impact on glucocorticoid responsiveness. Analysis with stageR detected 148 genes with a significant Article ll OPEN ACCESS treatment-genotype interaction, of which 34 showed an altered direction of significant change with glucocorticoid treatment ( Figure S3D). These data suggest that HNF6 is indeed less influential than HNF4A in shaping the response to glucocorticoid, with a lesser functional impact evident. By contrast, our data do support a role for HNF4A in shaping the tissue specificity of GR action. We suggest that, as a lineage-determining factor, HNF4A confers tissue specificity to the liver GR cistrome by maintaining chromatin accessibility at HNF4A motif-marked sites (assisted loading). In the absence of HNF4A, the regulatory landscape is remodeled, and GR binds to strong canonical GRE motifs normally inaccessible in the terminally differentiated hepatocyte.

DISCUSSION
In this study, we show that a substantial portion of the liver GR cistrome is characterized by HNF4A binding and the HNF4A motif. The presence of the HNF4A motif favors open chromatin, in comparison to sites where the HNF4A motif is not present. Strikingly, when HNF4A is removed, the chromatin environment and thus the GR cistrome are remodeled, with the HNF4A motif enriched at sites where GR binding is lost. Furthermore, GR binding emerges at sites where GR is typically bound in non-liver tissues where chromatin is not normally accessible in liver. At these sites, we see enrichment of FOX factor motifs, notably FOXM1, whose gene expression is also upregulated in Hnf4anull liver.
Multiple previous studies have demonstrated the presence of the HNF4A motif at GR binding sites (Caratti et al., 2018;Hemmer et al., 2019;Lim et al., 2015;Quagliarini et al., 2019), and have shown the tissue specificity of nuclear receptor cistromes (Gertz et al., 2013;John et al., 2011). C/EBPB and the basic-helix-loop-helix factor E47 have also been shown to play important roles in regulating hepatic glucocorticoid action Hemmer et al., 2019). This study builds on these works by directly comparing the GR cistrome in Hnf4a-intact and Hnf4anull liver, identifying those GR binding sites that are dependent on HNF4A, and showing that loss of tissue specificity extends to the emergence of ''non-liver'' GR binding sites. Furthermore, we show that sites marked by the HNF4A motif, and those sites lost and gained in Hnf4a-null liver, have distinct profiles of chromatin accessibility.
The characteristics of the GR sites that are gained and lost in the absence of HNF4A suggest a balance between chromatin accessibility (John et al., 2011) and GRE motif strength (Johnson et al., 2018) in specifying GR binding. Numbers of DNA-bound GR molecules per cell are thought to be in the order of the hundreds , and are thus outnumbered by the number of potential GR binding sites (motif analysis suggesting >137,000 GREs across the mouse genome). Where HNF4A maintains chromatin accessibility, GR may bind to a weak motif with considerable degeneracy from the canonical GRE. When HNF4A is not present, GR no longer binds these sites, but can instead bind strong GRE motifs at sites where chromatin is not normally accessible in liver ( Figure S4). GR has been proposed to have intrinsic pioneer function (Johnson et al., 2018), able to induce chromatin opening at inaccessible sites. Interestingly, our data are compatible with recent work on FOXA1 and HNF4A, which suggests that pioneer function is not a binary property of transcription factors but, rather, that TF binding is determined by its intrinsic physical properties and motif availability (Hansen et al., 2022). In a related fashion, major perturbations of the regulatory environment that likely induce chromatin remodeling (e.g., fasting [Goldstein et al., 2017], chronic high fat diet [Quagliarini et al., 2019]) have been shown to alter the observed actions of GR, and we suggest that, operating through a similar mechanism, this phenomenon extends to other nuclear receptors whose activity is state sensitive (Hunter et al., 2020;Zhang et al., 2017). Indeed, the ''cistromic plasticity'' of the estrogen receptor is proposed to be of clinical importance in breast cancer (Mayayo-Peralta et al., 2021).
While HNF4A and GR have been identified together in ChIP-MS studies (Quagliarini et al., 2019), our data suggest a permissive role for HNF4A akin to what is proposed for C/EBPB-that of maintaining chromatin accessibility at commonly occupied sites-rather than direct co-operative interaction between the two nuclear receptors. When HNF4A is lost, we see greatly reduced chromatin accessibility at HNF4A-marked sites. Recent complementary recent work has also demonstrated loss of active histone marks H3K4me1 and H3K27ac in Hnf4a fl/fl Alb Cre LKO liver (Qu et al., 2021). In addition, there is a broad distribution of inter-motif distances, with many GRE-HNF4A motif pairs lying further apart than the 20 bp proposed for high-confidence co-occupancy (and thus physical co-operativity) (Sö nmezer et al., 2021). There are clearly many sites where GR binding is not dependent on HNF4A, and more dynamic context specificity of GR action will also be conferred by the ultradian and circadian variation in the availability of its endogenous ligand (Conway-Campbell et al., 2012;Ince et al., 2019). Thus, the combinatorial actions of lineage-determining factors, state-sensitive factors or chromatin remodeling enzymes, and the rhythmicity of its ligand, confer exquisite context specificity to GR action, and must be taken into account when considering therapeutic applications.
Limitations of the study This study demonstrates, in vivo, the remodeling of the GR cistrome with the deletion of a lineage-determining factor. Our data echo the results of previous tissue-tissue comparisons of nuclear receptor binding (Gertz et al., 2013), but now show directly the importance of a single factor. This value of the study is inextricably linked to a confounding factor, that of the abnormal liver function that results from disruption of HNF4A expression. The livers of Hnf4a fl/fl Alb Cre LKO mice are enlarged, demonstrating hepatocyte hypertrophy and lipid accumulation (Hayhurst et al., 2001). This makes it difficult to perform more detailed physiological studies in this line. However, we mitigated the liver pathology as much as possible by studying animals at a young age, in what is a widely used mouse line (Fekry et al., 2018;Karagianni et al., 2020;Wu et al., 2020;Xu et al., 2021). Sites where GR binding is lost enrich most strongly for the HNF4A motif (this is also the case for sites of reduced chromatin accessibility), and overlap with HNF4A binding sites, supporting an effect specific to HNF4A loss, rather than attendant liver pathology. Undoubtedly, however, some remodeling of the GR cistrome will reflect indirect rather than direct effects of Hnf4a 10 Cell Reports 39, 110697, April 19, 2022 Article ll OPEN ACCESS deletion (e.g., those sites that do not overlap with the HNF4A cistrome; Figure 2F). This is a caveat of using this in vivo model. Unfortunately, alternative approaches using primary cell culture are difficult, due to rapid loss of differentiated cell function, and liver cancer cell lines offer poor models of liver biology. Similarly, the large impact of Hnf4a deletion on the liver transcriptome makes it challenging to distinguish which gene expression changes are specifically due to the loss of HNF4A regulation of GR action. We have taken a statistical association approach (Briggs et al., 2021) to link remodeled GR sites to genes with altered DEX response, which does not prove cause-effect, but does have the advantage of incorporating thousands of genomic loci, in an unbiased manner. Cause and effect might be inferred by mutating or deleting putative enhancers in a hepatic cell line. However, testing the number of remodeled GR sites we detect would require a massively parallel reporter assay (testing a smaller number of sites might introduce selection bias), and we cannot be certain that the chromatin environment in an artificial system would be representative of that in vivo. Thus we limit our conclusions to the likelihood that the remodeling of the GR cistrome is of functional importance.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
The authors declare no competing interests.

INCLUSION AND DIVERSITY
One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. While citing references scientifically relevant for this work, we also actively worked to promote gender balance in our reference list.
Article ll OPEN ACCESS